Astroviruses (AstV) are single-stranded, positive-sense RNA viruses, best known for causing diarrhea in humans and are also found in many other mammals; in those, the relevance in gastroenteritis remains unclear. Recently described neurotropic AstV showed associations with encephalitis in humans as well as in other mammals. In Switzerland, two different neurotropic AstV were identified in cattle, as well as one in a sheep. The high genetic similarity between the ovine and one of the bovine AstV strengthens the hypothesis of an interspecies transmission. In humans, AstV associated with encephalitis were found also in human stool samples, suggesting that in these patients the infection spreads from the gastrointestinal tract to the brain under certain conditions, such as immunosuppression. Whether a similar pathogenesis occurs in ruminants remains unknown. The aims of this study were (1) the investigation of the potential occurrence of neurotropic AstV in feces samples, (2) the discovery and analysis of so far unknown AstV in small ruminants and other ruminant species’ fecal samples and (3) the examination of a potential interspecies transmission of AstV. To achieve these aims, RNA extraction out of 164 fecal samples from different ruminant species was performed and all samples were screened for known neurotropic AstV occurring in Switzerland, as well as for various AstV using RT-PCR. Positive tested samples were submitted to next generation sequencing. The generated sequences were compared to nucleotide- and amino acid databases, virus properties were identified, and phylogenetic analyses as well as recombination analysis were performed. The excretion of neurotropic AstV in small ruminants’ feces could not be demonstrated, but this work suggests the first identification of AstV in goats as well as the discovery of multiple and highly diverse new genetic variants in small ruminants, which lead to a classification into novel genotype-species. Additionally, the prediction of multiple recombination events in four of five newly discovered full or almost full-length genome sequences suggests a plausible interspecies transmission. The findings point out the occurrence and fecal shedding of previously unknown AstV in sheep and goats and pave the way towards a better understanding of the diversity and transmission of AstV in small ruminants. Astroviruses (AstV) are non-enveloped, single-stranded positive-sense RNA viruses with an icosahedral virion structure, appearing as a star-like shape in electron microscopy . The AstAccording to the affected host class, two AstV genera were established: Mamastroviruses (MAstV) representing genotype species affecting mammalian species and Avastroviruses containing those viruses found in avian species. Due to the availability of high throughput next-generation-sequencing (NGS) technologies and the use of broadly reactive Pan-AstV RT-PCR protocols, there has been a remarkable increase in the number of AstV discovered in diverse species during the last years . AstV weIn 2010, AstV were found for the first time in association with encephalitis in a child with immunodeficiency . ThereafIn animals, the state of knowledge about the tissue tropism of AstV is even more limited. Even though the presence of ovine astroviruses (OvAstV) in fecal sheep samples constituted the first report of AstV in animals , still lSince 2013, different novel AstV genotype species were found as a plausible cause of non-suppurative encephalitis in cattle  and a feTo date, there are 19 genotype species of Mamastrovirus (MAstV 1–19) recognized by the International Committee on Taxonomy of Viruses (ICTV). In particular, in OvAstV, little is known about their diversity. OvAstV-1 belongs to MAstV 13 and is the only enterotropic AstV closely related to neurotropic strains, but their exact taxonomy is still pending . Based oThe aims of the present study included the assessment of a potential shedding of neurotropic AstV in fecal samples, the investigation of diverse AstV in different ruminant species and the examination of a potential interspecies transmission. Fecal samples of sheep, goats, deer, alpaca, and llamas were tested for BoAstV-CH13 as well as BoAstV-CH15/OvAstV-CH16 and screened for other AstV using a Pan-AstV RT-PCR. NGS and bioinformatics were used to recover viral genome sequences and to perform a phylogenetic comparison as well as a recombination analysis with other known AstV.Fecal samples submitted for parasitological diagnostics were kindly donated by the Institute of Parasitology, Vetsuisse Faculty, University of Bern . In total 164 fecal samples, derived from 56 sheep, 56 goats, 30 alpacas, 12 deer, four llamas, two bisons, two chamois, one giraffe, and one ibex were investigated. Additional information on the animals and their health status was not available. Fecal samples were suspended 1:10 (w/v) in sterile PBS  and stored at −80 °C until further analysis. BoAstV-CH13 and OvAstV-CH16 positive and negative brain tissues were used as positive and negative controls for RT-PCRs and were available from the archive of the Division of Neurological Sciences, Vetsuisse Faculty, University of Bern . The summary of the laboratory workflow is shown in g for 3 min. The supernatants were then centrifuged through Vivaclear MINI Clarifying filters with 0.8 μm PES  at 2,000×g for 5 min. Next, 280 μL of the filtrates were treated with 2 μL Benzonase (1U/μL)  for 2 h at 37 °C. The benzonase was inactivated by adding ethylenediaminetetraacetic acid (EDTA) to a final concentration of 5 mM. Finally, RNA extraction was performed with the QIAamp Viral RNA Mini Kit  with the modification that the carrier RNA was omitted. RNA extracts were stored at –80 °C until further analysis.All RNA extractions from fecal suspensions for AstV-screening by RT-PCR were done with the QIAamp Viral RNA Mini Kit  and RNA isolation from brain tissue was performed with TRI Reagent  according to the manufacturers’ protocols. To prepare samples for NGS, 500 μL of fecal suspensions were centrifuged at 16,000×To detect BoAstV-CH13, the RNA extracts were analyzed by either of two different probe based RT-qPCR assays (CH13-A or CH13-B) as described previously. While the CH13-A assay targets the 5′ part of the viral genome in ORF1a, the CH13-B assay detects the center of the viral genome in ORF2. Both protocols revealed a very similar excellent accuracy, precision, and analytical sensitivity .For the detection of BoAstV-CH15 as well as OvAstV-CH16 a primer pair and a probe were designed based on full-genome sequence alignments of the following three strains from Switzerland and Germany; BoAstV-CH15, OvAstV-CH16 and BoAstV-BH89/14, targeting the 3′ end of ORF2 using the Geneious software package . The RT-qPCR was performed as described previously . After eFor the detection of other AstV, a previously described heminested RT-PCR protocol using five degenerated primers; four forward and one reverse primer, which target a 450 nt long sequence at the 3′ end of ORF1b of a broad panel of AstV was used . Primer Prior to NGS, libraries were prepared using TruSeq DNA Nano Kit . For cDNA synthesis and polyA-selection the SMARTer Ultra Low Input RNA Kit  was applied. NGS was performed on an Illumina HiSeq 3000 or NovaSeq 6000 in paired-end mode (2 × 150 bp).PRJNA30567, assembly Vicugna_pacos-2.0.1, deer: BioProject PRJNA324173, assembly CerEla1.0, goat: BioProject PRJNA340281, assembly ARS1, sheep: BioProject PRJNA179263, assembly Oar_v4.0) using STAR (Version 2.5.3a). Quality-trimmed and unmapped reads were assembled via SPAdes (Version 3.11.1).Reads were quality-trimmed with trimmomatic (Version 0.36) and mapped to their respective host genomes  using BLASTn  and DIAMOND . In order to exclude false positives, the scaffolds with a virus hit were aligned to an in-house non-viral database consisting of archaeal, bacterial, fungal, mammal, and protozoal sequences. Scaffolds were considered false positive if they had a longer hit on a sequence of the in-house database compared to the virus databases or if they had a nucleotide hit of more than 10% of their own length to any sequence of the non-viral database. Scaffolds with hits to AstV on nucleotide and/or amino acid level were considered valid AstV hits. For further analysis, scaffolds with a length over 5,550 nt and the three AstV-typical ORF including the ribosomal frameshift sequence between ORF1a and ORF1b were selected.In order to complete the inchoate 5′ ends of the five almost full-length scaffolds, a rapid amplification of cDNA ends (RACE) was performed. This 5′ RACE was carried out with a RACE-System  according to the manufacturer’s instructions except of the usage of SuperScript™ III Reverse Transcriptase  instead of SuperScript™ II Reverse Transcriptase . Nested PCR was performed with gene specific primers  and Taq After visualization on a 1% agarose gel, PCR-products were excised of the gel and purified using NucleoSpin Gel and PCR Clean-up system  according to the manufacturer’s protocol.® Terminator v3.1 Cycle Sequencing Kit  in a 3730 DNA Analyzer  according to the manufacturer’s protocol. Sequence data were analyzed using the Geneious software package , trimmed with an error probability limit of 0.05 on both ends and aligned to the respective scaffolds.Each 3 μL of the purified PCR products was Sanger-sequenced using the BigDyeCapsid precursor sequences of 36 AstV strains were used for phylogenetic analysis and included AstV strains of various ruminants and our newly discovered sequences. Based on these sequences, a phylogenetic tree was constructed using the maximum likelihood method with 1,000 bootstrap replicates based on the Le_Gascuel_2008 with Freqs. model  in MEGA http://web.cbio.uct.ac.za/∼darren/RDP4Manual.pdf) and with the highest acceptable p-value set to 0.01. Recombination events were considered only when involving at least one of our newly generated sequences and having a highest acceptable p-value of 0.01 with all of the following methods: RDP  , followiods: RDP , GENECONods: RDP , Bootscaods: RDP , Maxchi ods: RDP , Chimaerods: RDP , SiScan ods: RDP  and 3Seqods: RDP .To confirm novel AstV sequences, discovered by NGS, a RT-qPCR using specific primer-probe-combinations for each putative virus-candidate was applied. Specific primers and probes were designed for each of the almost full-length scaffolds using the Geneious software package . The primer and probe sequences with the respective nucleotide positions of the target are provided in All RT-qPCR reactions were performed using the TagMan™ Fast Virus 1-Step Master Mix  in 10 μL reactions according to the manufacturer’s instructions with a final concentration of 500 nM for each primer and 125 nM for each probe. For each reaction 2 μL RNA were added to 8 μL Master-Mix. The RT-qPCR was performed using a CFX96™ Real Time System on C1000 Touch™ Thermal Cycler , with the following cycle settings: 10 min, 45 °C; 10 min, 95 °C, and 40 cycles . After each elongation step, fluorescence was measured and analyzed with the CFX Maestro software  with an auto calculated baseline threshold. Samples with a ct-value <35 were defined as positive.After confirmation of all newly discovered AstV-candidates, all remaining 159 samples were re-tested using the RT-qPCR protocols as described above. For each run, a non-template control was used as negative control. The respective confirmed samples served as target-specific positive controls.Quantitative RT-PCRs were performed for the detection of neurotropic AstV. Positive controls showed ct-values on average of 22.73 (22.62–22.86) for BoAstV-CH13 and 23.22 (21.74–24.61) for OvAstV-CH16, respectively. All negative controls were scored negative. All 164 fecal samples had ct-values >35 or were reported as “undetectable” and were therefore diagnosed as negative for BoAstV-CH13 and OvAstV-CH16.n = 6: S1–S6), goats (n = 8: G1–G8), alpaca  and deer  showed a band at 450 bp and were therefore defined as Pan-AstV RT-PCR-positive.To ensure the effective implementation and to establish positive controls, the protocol was first applied to brain-extracts of BoAstV-CH13 and OvAstV-CH16 positive animals. These samples showed a strong and clear band with the expected size of 450 bp. Nineteen of the 164 tested fecal samples derived from sheep . In 12 samples, a total of 29 scaffolds ranging from 521 to 6255 bp in size, showed AstV hits in the bioinformatics analysis on amino acid level, with sequence identity to known AstV sequences ranging from 39.3% to 96.4% (Sequencing in paired-end mode (2 × 150bp) generated 45′455′879–233′701′511 reads per sample and raw reads were deposited in the European Nucleotide Archive , CapAstV-G3.1 (MK404646.1), CapAstV-G5.1 (MK404647.1), OvAstV-S5.1 (MK404648.1), and OvAstV-S6.1 (MK404649.1)).In four of the five samples analyzed, the 5′ RACE did work and Sanger sequencing added to the 5′ end of the respective almost full AstV scaffolds . This resulted in the completion of these four scaffolds, which, together with CapAstV-G2.1, were deposited on GenBank (Accessions: CapAstV-G2.1 (p-dist. 0.293) and BoAstV-B170/HK (p-dist. 0.361), respectively. CapAstV-G5.1 is very similar to the new OvAstV strains S5.1 (p-dist. 0.236) and S6.1 (p-dist. 0.027), clustering together in one clade of the phylogenetic tree. They cluster with a p-distance of 0.419 to the closest related AstV strain CcAstV-1/DNK/2010, which has been detected in feces from deer  for the primer-probe-pair S6.1, which is not surprising, considering the fact that these two samples share highly similar sequences. In all other samples, no cross-reactivity could be detected between the individual targets.All 159 samples remained negative for the primer-probe combinations G3.1, G5.1, S5.1, and S6.1. Surprisingly one sample originating from a goat, scored positive for G2.1 . Based on information from the original submission site , it could be found that this sample originated from the same holding as the sample G2.This study presents the discovery and molecular characterization of putatively new AstV genotype species in sheep as well as in goats. These novel AstV show a broad genetic diversity compared to AstV affecting other species. The present results support interspecies transmission of AstV between goats and sheep, as well as recombination events between AstV affecting sheep, goats and cattle.The aims of the study were to investigate the potential shedding of neurotropic AstV in small ruminants’ feces, to discover and to analyze unknown AstV, and examine a putative interspecies transmission. Therefore, all available samples first were screened for the neurotropic AstV BoAstV-CH13 as well as BoAstV-CH15/OvAstV-CH16. Three different methods were then applied, first, highly specific RT-qPCR, second, a wide-spectrum RT-PCR, and third, NGS of all samples interpreted as positive after RT-PCR. None of these samples could be defined as positive for BoAstV-CH13 or BoAstV-CH15/OvAstV-CH16. Thus, there is no evidence that neurotropic AstV are part of the intestinal virome of small ruminants. Noteworthy, limitations of this study are the relative small number of samples investigated, the unknown disease status of the animals and the focus mainly on sheep and goats due to a lack of available samples from other ruminant species. A related study targeting feces and brain of cattle was reported previously .−3 mutations per nucleotide per genomic replication  had its best hit on the ORF2 region of BoAstV-CH13  and clusFor the remaining 23 scaffolds, best hits were consistently to bovine strains and, unexpectedly, not to previously described ovine (OvAstV-1 and OvAstV-2)  and deerFive novel, including four full-length and one almost full-length AstV sequences were discovered: two in sheep and three in goats. Noteworthy, this study reports for the first time an AstV infection in goats. Based on the genetic distances of the capsid protein precursor sequences to the closest related strains, these caprine and OvAstV are grouped into three different genotype species. According to the ICTV 9th report 2011 , amino aA retrospective screening of the whole sample panel targeting the five newly discovered AstV candidates resulted in only one additional positive sample for target G2.1. This sample originated from the same holding as animal G2 and was collected at the same day. While this screening indicated an overall low prevalence of the newly discovered AstV candidates, it appears plausible that these viruses transmit between animals and that the in herd prevalence may be much higher.Astroviridae was already challenged in previous studies (Because recombination between viruses requires infection of the same host cell, the existence of interspecies transmission events are further supported by predicted recombination events . In the  studies .The question whether enterotropic AstV can cause disease in small ruminants remains so far unresolved. Due to the mainly unknown health status of the tested animals, the importance of AstV occurring in small ruminants’ feces remains so far unclear and needs to be further investigated. Still, this study describes five novel AstV discovered in small ruminants, including the first description of an AstV in goats and gives new insights into the frequency and diversity of AstV in ruminant species.10.7717/peerj.7338/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.7338/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.7338/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.7338/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.7338/supp-5Supplemental Information 5Click here for additional data file.


A strategy is devised to synthesize zwitterionic acetylated cellulose nanofibrils (CNF). The strategy included acetylation, periodate oxidation, Schiff base reaction, borohydride reduction, and a quaternary ammonium reaction. Acetylation was performed in glacial acetic acid with a short reaction time of 90 min, yielding, on average, mono-acetylated CNF with hydroxyl groups available for further modification. The products from each step were characterized by FTIR spectroscopy, ζ-potential, SEM-EDS, AFM, and titration to track and verify the structural changes along the sequential modification route. The scope of application of nanocelluloses, including cellulose nanofibrils (CNF), microfibrillated cellulose (MFC), and cellulose nanocrystals (CNCs) , may be d-anhydroglucopyranose units (AGUs). Covalent modification of nanocellulose, including oxidation, esterification, etherification, grafting copolymerization, and ring-opening reactions, has recently been the subject of several comprehensive reviews .,32.3−) i−1. Since aldehydes introduced by periodate oxidation also contain C=O, a band at approximately the same wavenumber is expected for ACNF-Ox. When the spectra are baseline corrected and normalized, however, the relevant peaks in both samples can be compared based on the energy absorbed at the different regions. A clear decrease in absorption is then observed for the OH band around 3340 cm−1 for ACNF-Ox compared to ACNF, while the intensity of the carbonyl band is increased. This indicates that some of the secondary OH groups have undergone oxidation to aldehyde groups. This is further sustained by a decrease in intensity of the secondary alcohol band at 1105 cm−1 for ACNF-Ox (FTIR spectra of ACNF and oxidized ACNF (ACNF-Ox) are quite similar . Acetyla ACNF-Ox . ACNF-Ox were titrated with hydroxylamine hydrochloride and the average number of aldehyde groups was quantified according to Equation (3). The calculated extent of oxidation was 34%. A linear polymer-like structure could possibly be obtained at certain regions of CNF as the fibril structure is lost due to ring-opening. The crystal structure gets increasingly disrupted as the rings open up due to a significant reduction in hydrogen bonds. In this work, we targeted a limited degree of aldehyde content in order to preserve the nanofibrillar and crystal structure of CNF as much as possible.The ζ-potential of ACNF-Ox was −33.7 ± 0.23 mV , which iN,N-dimethyl-1,3 propanediamine (Aminated ACNF (ACNF-Am) were synthesized from ACNF-Ox and ediamine . The priediamine . The aciAfter completion of the Schiff base reaction (6 h), we added an aqueous sodium borohydride solution to the reaction mixture to reduce the double bond in the Schiff base groups to secondary amine groups, ACNF-Am . This st−1. This band overlaps with the broad OH band in the 3340 cm−1 region, which makes it very difficult to distinguish one band from the other. When the spectra are baseline corrected and normalized, however, it is clear that there is a rather large increase in absorption intensity for ACNF-Am around 3340 cm−1, compared to ACNF and ACNF-Ox  by reacting ACNF-Am with sodium 2-bromoethanesulfonate (SBES) . The rea−1 region, but due to overlap, this band is poorly resolved. Still, there is an increase in absorption at 1161 cm−1 which could be attributed to the sulfonate symmetric S=O stretch. An increase is also observed at 1369 cm−1, which could be assigned to the sulfonate antisymmetric S=O stretch. Weak quaternary ammonium N–C stretches can usually be detected at 1639–1645 cm−1 [FTIR spectra of ACNF-Am and ACNF-Zwi are quite similar . The int645 cm−1 . Water bThe elemental composition of ACNF-Zwi was determined with SEM-EDS to further investigate if the quaternary ammonium formation reaction was successful. If so, the presence of sulfur will indicate the presence of sulfonate groups in the structure. The SEM-EDS instrument we used does not accurately detect hydrogen and the atom numbers given for light elements (atom numbers < 10), such as oxygen, nitrogen, and carbon, are highly approximate. Still, sulfur is measurable and was indeed detected in ACNF-Zwi (1.5 ± 0.2%). An EDS spectrum is available in the SEM and AFM images of CNF and the produced ACNF and ACNF-Zwi are available in the The sequential modification scheme for CNF described here was elaborated and investigated in an effort to broaden the library of CNF derivatives and potentially open up for new applications, where CNF is currently not considered a viable component. Nanocellulose has many interesting, and potentially valuable, properties and is often put forth as a high-performance component candidate in future, sustainable materials, not in the least thanks to their inherent renewability ,4,5,6,7.Here we tried to take the above into account throughout the functionalization route. The first step, mono-acetylation, was introduced to alter the hydrophilicity. The reaction was performed in glacial acetic acid, considered a more eco-friendly solvent than many other solvents employed for acetylation of cellulose . There iThe first protocol for periodate oxidation of ACNF used DMSO as a medium, nitrogen as an atmosphere and allowed the reaction to occur for 6 h at 45 °C . The proThe final step most likely produced zwitterionic CNF with quaternary ammonium groups. Such functionalities open up new avenues of possible applications of CNF. Quaternary ammonium cations are permanently charged species at a variety of different pH values and are unreactive toward oxidants, acids, electrophiles, and nucleophiles. The hydrophilic structure of ACNF-Zwi could be beneficial for future use in water filter systems as the membranes are more easily hydrated which could simplify interactions between the zwitterionic groups and heavy metal contaminants in water effluents . As quatN,N-dimethyl-1,3-propanediamine 99%, sodium borohydride, sodium acetate ≥99% and sodium 2-bromoethanesulfonate 98% (SBES) and hydroxylamine hydrochloride ≥99.9995% were purchased from Sigma-Aldrich . Acetic anhydride ≥99.0% was purchased from Fluka Chemika . Glacial acetic acid 99.79% was purchased from Fisher Scientific . Dimethyl sulfoxide ≥99% (DMSO) was purchased from Merck . Hydrochloric acid (HCl) was purchased from J.T. Baker . Absolute ethanol and sodium hydroxide (NaOH) 99.3% were purchased from VWR Chemicals . Microfibrillated cellulose paste (Exilva P01-V) with a solid content of 10.1% (w/w) was a kind gift from Borregaard . Exilva P01-V has a viscosity (2% in H2O) of 23,000 mPas and a conductivity (2% in H2O) of 23 μS/cm. Deionized (DI) water was used throughout the study. All chemicals were used without further purification.Sulfuric acid 95–98%, acetone, sodium (meta) periodate, ethylene glycol ≥99%, The CNF paste  was suspended in DI water (200 mL) in a round-bottomed flask (500 mL) and stirred until the nanofibers were thoroughly dispersed in the aqueous phase. Then, the suspension was probe sonicated  using a CV334 probe sonicator from CIAB Chemical instruments AB with a maximum input power of 750 W and a maximum frequency of 20,000 times/s at an amplitude of 20%. Afterward, the suspension was put in liquid nitrogen, under constant rotation, until the mixture became completely frozen. The frozen suspensions were then lyophilized using a SCANVAC CoolSafe freeze dryer with a vacuum of 0.250 hPa and a condenser temperature of −82 °C for 72 h.The acetylation reaction was developed by modification of a previously described protocol  to reducPeriodate oxidation was explored via two different protocols: One performed in DMSO and one using water as the reaction medium. ACNF suspension was solvent exchanged from water to DMSO according to a procedure described elsewhere . BrieflyThe oxidation was then carried out in DMSO and in the dark . The solA previous protocol for periodate oxidation  was modiN,N-dimethyl-1,3-propanediamine  was added under a nitrogen atmosphere. The reaction mixture was heated to 45 °C and the mixture was left to react for 6 h. The reaction slowly turned a faint yellow color which turned darker with time. Next, 2 equivalents (with respect to ACNF) of 5% (w/v) sodium borohydride aqueous solution were added slowly to the mixture through a syringe. The mixture was left to react, under the same conditions, for an additional 3 h. The reaction was terminated by the addition of a large excess of ice-cold DI water. The mixture was washed by centrifugation , using an Avanti J-E centrifuge from Beckman Coulter, and re-dispersion with ice-cold DI water. This washing procedure was repeated 8 times before the product was acquired by collecting the pellet. The resulting product is an aminated CNF, herein referred to as ACNF-Am. The moisture content of ACNF-Am was determined by drying a known amount of product in an oven at 60 °C for 24 h. The obtained ACNF-Am was kept at 4 °C until further use.ACNF-Ox  was dispersed in acetate buffer  mixed with 500 mL of acetic acid (0.2 M)) according to a previous protocol . The mixACNF-Am was solvent exchanged from water to DMSO as described in The moisture content of each modified CNF sample was determined by drying a known amount of sample in an oven at 60 °C for 24 h and recording the weight loss. −1 in the spectral region of 4000–600 cm−1. Corrections were made for atmospheric carbon dioxide and water. All spectra were baseline corrected and normalized at the C–O stretch band at 1030 cm−1 originating from the cellulose backbone [FTIR spectra of all obtained products were recorded at room temperature using a PerkinElmer Spectrum 2000 FTIR, equipped with an ATR Golden Gate from Graseby Specac . All spectra were acquired through 64 scans with a spectral resolution of 4 cmThe obtained products were suspended in ice-cold DI water. The concentration of each sample corresponded to 0.3 mg (dry weight)/5 mL. The pH of each sample was in the range of DI water. The samples were immersed in dry ice and then probe sonicated  using a CV334 probe sonicator from CIAB Chemical instruments AB with a maximum input power of 750 W, and a maximum frequency of 20,000 times/s at an amplitude of 20%. The samples were then stirred for 30 min before the ζ-potential was measured, using a Zeta potential Zetasizer Nano ZS from Malvern Instruments. Three replicates were carried out for each sample to calculate an average ζ-potential and standard deviation. CNF, ACNF, and ACNF-Zwi were oven-dried overnight at 60 °C. The dried samples were attached to sample supports with double-sided adhesive carbon tape and sputter-coated with a thin layer (~8 nm) of Pt/Pd under an inert atmosphere using a Cressington 208 HR. The samples were then analyzed by ultra-high-resolution field emission scanning electron microscopy (FE-SEM) using a Hitachi S-4800 operating at 5 kV. The elemental composition of each sample was then determined with an X-MaxN from Oxford Instruments. All samples were analyzed at three different regions. For ACNF and ACNF-Zwi, the average weight percent (wt. %) and standard deviation were calculated through the data obtained from these three different areas. The degree of substitution for acetyl groups in ACNF was quantified using a protocol described elsewhere . BrieflyThe degree of substitution of aldehyde groups in ACNF-Ox was quantified using a protocol described elsewhere . ACNF-OxThe cellulose nanofibers and the modified versions were examined using AFM to understand the morphology of the materials. The samples were prepared by dispersing the freeze-dried nanofibers in water and drop-casting from dilute suspension on mica. An atomic force microscope (AFM) Nanoscope V from Veeco Instruments Inc., Plainview, NY, USA, at a resonance frequency of 310 kHz was used and the nanofiber sizes were evaluated from the height images using Nanoscope V software.A sequential modification, including acetylation, periodate oxidation, Schiff base reaction, and borohydride reduction, as well as a quaternary ammonium reaction was devised to prepare zwitterionic acetylated CNF. The presence of the acetyl groups was verified via FTIR spectroscopy, titration, and ζ-potential. The extent of acetylation was determined to 30% via titration, thus mono-acetylated CNF (ACNF) was obtained. The occurrence of aldehyde groups in oxidized ACNF was established via FTIR spectroscopy, titration, and ζ-potential. The extent of oxidation was determined as 34%; a higher degree of oxidation was not desired in order to preserve the fibril structure and crystallinity of the material to a larger extent. The aldehyde groups were used as auxiliaries to introduce secondary amine groups to the structure, as indicated via FTIR spectroscopy and ζ-potential measurements. Lastly, quaternary ammonium cations and sulfonate anions were introduced to the structure. The end product was analyzed via FTIR spectroscopy, ζ-potential, AFM, and SEM-EDS.


Bantam is a conserved miRNA highly expressed in insects. We previously showed that the antisense inhibitor (antagomiR) of bantam improved the infection by baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) in Spodoptera exigua and S. litura larvae. Here, we constructed a recombinant AcMNPV (vPH-banS) expressing bantam sponge, an mRNA containing eight antisense binding sites for bantam. Infection with wild type AcMNPV (WT) or the control recombinant virus vPH resulted in a significant increase of bantam level, whereas infection with vPH-banS led to an approximately 40% reduction of bantam in both Sf9 cells and S. exigua larvae. Although, comparable production of budded virus and polyhedra were detected in vPH-banS-, vPH-, and WT-infected Sf9 cells, vPH-banS showed remarkably increased insecticidal activity in S. exigua larvae. The 50% lethal concentration and the median lethal time of vPH-banS was only 1/40 and 1/2, respectively, of both vPH and WT. Further analysis showed that the level of molting hormone 20-hydroxyecdysone (20E) was significantly higher in larvae infected with vPH-banS than those infected with vPH or WT. This was confirmed by the result that the larvae treated with bantam inhibitor also had a markedly increased 20E level. Moreover, feeding larvae with 20E increased the virus-mediated mortality, whereas feeding with juvenile hormone partially reverted the high insecticidal effect of vPH-banS. Together, our results revealed that vPH-banS infection suppresses the level of bantam, and in turn elevates level of 20E in infected insects, resulting in increased susceptibility to baculovirus infection. Our study provided a novel approach to improve a baculovirus bio-insecticide by interfering with a key homeostasis-regulating miRNA of the host. Baculoviruses are a group of DNA viruses that infect insects and some other invertebrates . They arMicroRNAs (miRNAs) are small non-coding RNAs capable of regulating the expression of multiple target genes post-transcriptionally . They armiRNAs also play important roles in baculovirus infection and virus–host interaction. Virally encoded miRNAs have been found in baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) , Bombyx Bantam gene was first identified in the fruit flies Drosophila melanogaster and its overexpression caused the overgrowth of wings and eye tissues  supplemented with 10% FBS and antibiotics . The wild-type AcMNPV  and recombinant viruses vPH and vPH-banS (see below) were used in the study. The virus titer was determined by end-point dilution method in Sf9 cells. S. exigua (beet armyworm) larvae were reared individually in polymer cups on artificial diet , under the condition of 28°C, 70–90% humidity, and the photoperiod of 15L:9D.The fall armyworm polyhedrin gene was amplified from wild type AcMNPV DNA using the primers 5′-ttgaattctattttactgaattcgtaacagttttgt-3′ and 5′-tttctagagcacagaatctagagcttaataaatgta-3′ (EcoRI and XbaI sites are underlined) and cloned into EcoRI and XbaI-doubly digested pFsatbac-Dual , yielding pFBdual-PH. A bantam-sponge sequence, which contained eight repeats of bantam binding sequence  and restriction enzyme sites of NheI and XhoI at either end of the fragment, was chemically synthesized  and inserted into the two restriction sites downstream of the p10 promoter in pFBdual-PH, yielding pFBdual-PH-banS. Both pFBdual-PH and pFBdual-PH-banS were used to generate recombinant baculoviruses vPH and vPH-banS, respectively, using Bac-to-BacTM Baculovirus Expression System. The schematic diagram of the polyhedrin gene locus in these viruses is shown in Figure 1B. Recombinant viruses were confirmed by PCR using viral DNA extracted from the infected culture medium as the template and the primers 5′-gtgtttcagttagcctccc-3′ and 5′-gtgtttcagttagcctccc-3′.Recombinant baculoviruses were constructed using Bac-to-Bac system . The coding sequence of AcMNPV bantam in Sf9 cells, total small RNAs (<200 nt) were harvested from Sf9 cells and reverse-transcribed using miRVana microRNA Isolation Kit . RT-qPCR detection of bantam was performed using miScript II RT Kit  according to the manufacturer’s instructions. The forward primer of bantam and the internal control (U6 snRNA) were 5′-ctttctgagatcattgtgaaag-3′ and 5′-agagacgattagcatggccc-3′, respectively. The reaction was carried out on Stratagene MX3000p and data processing was done with the software MxPro. Relative expression of miRNA was normalized to U6 RNA by 2-ΔΔt method. Similar procedures were used to determine bantam expression in S. exigua insects, except that the larvae were first ground in liquid nitrogen.To determine the level of 2CO3-50 mM NaCl) for 2 h at room temperature. The suspension was then centrifuged at 2,600 × g for 15 min to remove undissolved debris. The supernatant (500 μl) was mixed with equal volume of lysate buffer , and 5 μl proteinase K (20 mg/ml), and placed at 50°C for 2 h. Viral DNA was extracted with phenol-chloroform and precipitated with ethanol. The amount of viral DNA was determined by qPCR using the primers targeting viral gp41 gene: 5′-agagttgggacagagcaacg-3′, 5′-gcgccaccgttgtaaaactt-3′. To determine the virus replication in insects, the infected larvae were ground in liquid nitrogen, and total DNA were extracted using QiaAMP DNA Mini Kit (Qiagen). The level of viral DNA was determined by qPCR as above, and normalized against host tubulin gene.Polyhedra were obtained from infected larvae and washed three times with PBS. The same amount of polyhedra of vPH-banS, vPH, and WT was left in alkaline solution  of each virus, polyhedra suspensions of different concentrations were sprayed on the surface of small pieces of diet to feed newly molted third instar larvae. New diet was added 24 h later when the contaminated diet was consumed completely. Mortality and pupation were recorded daily. Bantam antagomiR, a chemically modified antisense oligonucleotide of bantam, was designed, synthesized, and used to suppress the level of bantam in larvae as described previously .Larvae were weighted and phosphate buffered saline (PBS) was added to each larva according to the weight . They were then homogenized individually and centrifuged at 2 600 × bantam: 5′tagctttcacaatgatctca3′, mock: 5′cagtacttttgtgtagtacaa3′, synthesized and modified by GenePharma, Shanghai, China) in 100 μl hybridization buffer (prehybridization solution with 10% dextran sulfate) were applied per section, and incubated in a sealed humidified chamber for 16 h. Both prehybridization and hybridization were carried out at 27°C, 20°C below the Tm. After stringency washes, the slides were stained briefly with 6-diamidino-2-phenylindole (DAPI) for 5 min in dark. The samples were visualized using a Leica TCS SP8 epifluorescence microscope and LAS AF software .FISH experiment was carried out following a standard protocol . BrieflyPolyhedra were harvested from the larvae infected with vPH, vPH-banS, and WT, and washed three times with PBS. The polyhedra suspension was dropped on conductive tapes, and left at room temperature for 15 min. Then it was coated with a thin layer of gold and examined with a Hitachi TM3000 scanning electronic microscope.t-test. The showed results are representative of at least three independent experiments.Unless otherwise stated, bars represent means ± SD, and averages were compared using a bidirectional unpaired Student’s bantam inhibitor increased the insect mortality caused by baculovirus AcMNPV in both S. exigua and S. litura larvae  was inserted between every two binding sites . The sponge sequence was chemically synthesized and inserted downstream of the p10 promoter of the transfer vector pFastBac-Dual. The polyhendrin gene was inserted downstream of the polyhedrin promoter in the same vector. The recombinant baculovirus, vPH-banS , was obtained using the Bac-to-Bac system. A control recombinant virus with only polyhedrin gene downstream of the polyhedrin promoter (vPH) was also constructed . PCR and sequencing results confirmed the successful construction of vPH-banS and vPH .Our previous work showed that a larvae . To furtFigures 2A,B), though cells infected with vPH-banS had slightly higher level of polyhedrin mRNA and protein (data not shown). These results were consistent with our previous study using bantam inhibitor  production were observed for all three viruses , indicating that bantam sponge effectively suppressed bantam level in Sf9 cells.We further examined us study . HoweverS. exigua larvae and visualized using scanning electron microscope. As shown in Figure 3A, the polyhedra generated by the three viruses were similar in size and morphology. They also contained similar amount of viral DNA, as determined by qPCR , indicating that bantam sponge did not affect polyhedra morphogenesis.The polyhedra of vPH-banS, vPH, and WT viruses were isolated from infected S. exigua larvae were then inoculated with the same concentration of polyhedra (1 × 106polyhedra/ml) of vPH-banS, vPH, and WT, and the level of bantam was compared 4 d p.i. Similar to the results in Sf9 cells, infection with WT and vPH resulted in significant increase in bantam level, whereas infection with vPH-banS suppressed bantam level by about 40% compared to the mock-infected larvae .Third instar S. exigua larvae with polyhedra at 2 × 107 polyhedra/ml. As shown in Figure 4A, vPH-banS killed larvae much faster than WT and vPH, with the median lethal time decreased from 7.0 (WT) and 7.2 (vPH) days to 3.6 days . vPH-banS also had markedly reduced LC50, which decreased from 3.9 × 106  of vPH, and 4.9 × 106  of WT, to 1.2 × 105  polyhedra/ml of vPH-banS .The insecticidal activity of vPH-banS was further examined by infecting late second instar S. exigua larvae was further compared. Infection with vPH-banS showed lower body weight 5 d p.i., and lower polyhedra production than that in vPH and WT , whereas all three viruses had similar levels of viral DNA  5 d p.i. . These results suggested that the high infectivity of vPH-banS is mediated by its effect on host growth, rather than a change in virus replication.The replication of the three recombinant viruses in Drosophila, bantam was reported to repress the release of ecdysteroid hormone  and 20E level was measured 5 d p.i. As can be seen in Figure 5A, vPH-banS-infected insects had significantly higher level of 20E than those infected with WT or vPH .In  hormone . To exambantam antagomiR, a chemically modified antisense inhibitor of bantam miRNA . These data suggested that the increase of 20E after vPH-banS infection is related to the suppression of bantam.Further, second instar larvae were administrated with different concentrations of am miRNA , and 20Ebantam and 20E were measured in late second, third and fourth instar larvae before molting, and in newly molted early third, fourth, and fifth instar larvae. We found that bantam expression was high at the beginning of each instar, and low at the end of each instar, whereas the level of 20E followed an opposite pattern, which was low at the beginning of each instar, and high at the end of each instar . Much stronger fluorescence was also observed in the tissues of early fourth instar larvae than that of late third instar larvae when examined by fluorescent in situ hybridization using Cy3-labeled probe against bantam . These observations provided further evidence that bantam may also regulate 20E level in S. exigua.Next, the levels of bantam-mediated insecticidal activity, early third instar larvae were infected with vPH-banS and vPH (1 × 106 polyhedra/ml), together with 20E (16.25 μg/ml), or different concentrations of JH  in the diet. As shown in Figure 8, 20E supplementation further reduced insect survival to a similar degree in vPH-banS and vPH. Meanwhile, the addition of JH only slightly increased larval survival in vPH-infected larvae, whereas markedly increased larval survival in vPH-banS group. At 5 d p.i., the survival rate of vPH-banS-infected larvae increased from 39.8 to 73.6% with 250 μg/ml JH supplementation. At 7 d p.i., all of the larvae succumbed to vPH-banS infection, however, 250 μg/ml JH supplementation led to 26.8% survival in vPH-banS infected larvae. Since 20E and JH are thought to counteract with each other in regulating the growth and death of insect cells, the data support the notion that the elevated level of 20E accounted for the increased infectivity of vPH-banS.To further characterize the mechanisms underlying Bantam is a miRNA found specifically in insects and other invertebrates. Apart from Drosophila, bantam has also been reported in blood fluke Schistosoma japonica . It seems that the high expression level is important for bantam sponge to take effect.Apart from baculovirus Insect development is coordinately regulated by insect hormones, among which 20E and JH are of particular importance . As the bantam has been suggested to regulate host ecdysone hormone release in Drosophila (bantam antagomiR also markedly increased the levels of 20E. Thus, our data suggests that, as in Drosophila, bantam negatively regulates 20E level in S. exigua, though the exact mechanism is not clear and warrants further investigation.Since osophila , we furtegt, which can interact with 20E in the host. Expression of egt suppresses the level of 20E and extends the host survival so as to increase the production of progeny virus (bantam level after AcMNPV infection seems to have the same effect as egt, which could be another example of virus manipulating host physiology in favor of its own replication and dissemination.It has long been known that 20E has a role in baculovirus infection. Many members of baculoviruses encode the gene ny virus . The incbantam as an important host defending factor in response to baculovirus infection, and suppression of bantam by its sponge led to enhanced 20E level, thus contributing to the increased susceptibility to virus infection.The high level of 20E after vPH-banS infection is likely to be the most important factor for its high insecticidal activity. This was supported by the results that feeding larvae with 20E significantly increased virus-mediated larval mortality, whereas feeding with JH partially reduced the high mortality in vPH-banS group. Taken together, our data further demonstrated ie1 had increased transcriptional activity in the presence of 20E, though its effect on virus replication was not studied in vitro or in vivo. Alternatively, 20E might function through inhibiting cellular activity and inducing cell apoptosis. As the molting hormone, 20E binds to the nuclear receptor EcR, represses Myc level to inhibit the general insulin/IGF signaling, and limits cell proliferation and tissue growth (bantam might induce apoptosis by relieving bantam-mediated suppression of hid expression (Several mechanisms may account for the enhancement of virus infection by 20E. One possible reason is that 20E acts directly on virus infection. e growth . 20E hase growth . In addipression . Furtherpression . Howeverbantam modulates virus–host interaction through regulating the level of the molting hormone. Suppressing bantam led to the increase of 20E and resulted in increased mortality in virus-infected S. exigua. Baculoviruses are considered as bio-pesticides for agricultural pests. However, their slow mode of action has limited their utilization. Efforts have been made to improve their virulence. With further optimization, the bantam sponge-expressing recombinant baculovirus may have a great potential in the applications.In summary, we demonstrated that miRNA JZ and RL contributed conception and design of the study. ZR, XS, FH, JL, YOZ, YAZ, and JY performed the experiments. ZR, RL, and JZ wrote the manuscript. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus nematocida B16 and its host worm, the chemical 2-heptanone was found to be an important attractant for the hosts. So here we used this model to investigate the genes involved in synthesizing 2-heptanone in microorganisms. We identified a novel methylketone synthase gene yneP in B. nematocida B16 and found enhancement of de novo fatty acid synthesis during 2-heptanone production. Interestingly, a homolog of yneP’ existed in the non-pathogenic species Bacillus subtilis 168, a close relative of B. nematocida B16 that was unable to lure worms, but GC-MS assay showed no 2-heptanone production. However, overexpression of yneP’ from B. subtilis in both heterologous and homologous systems demonstrated that it was not a pseudogene. The transcriptional analysis between those two genes had few differences under the same conditions. It was further shown that the failure to detect 2-heptanone in B. subtilis 168 was at least partly due to its conversion into 6-methyl-2-heptanone by methylation. Our study revealed methylketone biosynthesis of Bacillus species, and provided a co-evolution paradigm of second metabolites during the interactions between pathogenic/non-pathogenic bacteria and host.Methylketones are broadly distributed in nature and perform a variety of functions. Most microorganisms are thought to produce methylketone by abortive β-oxidation of fatty acid catalytic metabolism. However, two methylketone synthetase genes in wild tomatoes are reported to synthesize methylketone using intermediates of the fatty acids biosynthetic pathway. In our previous study on Trojan horse-like interactions between the bacterium  Lactobacillus casei (Staphylococcus carnosus produces 2-pentanone and a highly autolytic strain of Lactobacillus helveticus is reported to enhance the levels of methylketones during cheese ripening (5–C11) are aromatic substances, commonly enriched in essential oil from leaves and fruits; while medium-chain methylketones (C7–C15) provide resistance to insects  of us casei . Staphylripening . In fungripening . In plan insects . In anim insects . Some sm insects . A highe females .Penicillum roqueforti catalyzed the production of 2-undecanone, 2-heptanone, and 2-pentanone from short-chain alkyl esters of lauric acid, octanoic, hexanoic in an aqueous-organic two-phase system, respectively (Solanum habrochaites) that could produce methylketones, the gene expressional levels involved in fatty acid synthesis were much higher than those of fatty acid beta oxidation pathway , which was one of the intermediate products in fatty acid synthesis, into the products of methylketone  seeded with E. coli OP50. Then the well-fed adult C. elegans were prepared by washing three times with M9 buffer and once with water for the following experiments.The strains and plasmids used in this study are listed in yneP and RyneP for the yneP gene, FytpA and RytpA for the ytpA gene in B. nematocida B16, and FyneP’ and RyneP’ for the yneP’ gene in B. subtilis 168. Those DNA fragments obtained from gel recovery were ligated to the pMD-19T vector respectively, and then the recombinant plasmid was transformed into E. coli DH5α competent cells. After the positive transformants were validated by PCR reactions and DNA sequencing, they were digested with BamHI and XohI at the primer-incorporated restriction sites, and then inserted into a BamHI/XohI digested pET28a vector to obtain the recombinant plasmids pET28a-yneP, pET28a-ytpA, and pET28a-yneP’. Those three recombinant plasmids were finally transformed into the competent cells of E. coli BL21 again to express the target proteins.To confirm the functions of candidate genes in methylketone synthesis, the full opening reading frames (ORF) of target genes were amplified via PCR using the primers of Exp FyneP’ in its native host B. subtilis 168, the yneP’ ORF from B. subtilis 168 was amplified using primers FbsyneP’ and RbsyneP’ that had the primer-incorporated restriction sites of SalI and SphI. The vector pDG148 was used to construct the recombinant plasmid pDG148-yneP’ and this recombinant plasmid was finally transformed into competent cells of B. subtilis 168.In order to over-express Selected positive transformants were cultured by incubation overnight at 37°C with shaking in 5 ml of LB supplemented with 50 μg/ml kanamycin. After a 3 ml overnight culture was transferred into 300 ml of fresh LB medium and grown until an OD600 reached 0.3, 0.1 mM IPTG was then added to induce the protein expression for 4 h. The supernatant was discarded by centrifugation at 8000 rpm for 10 min. The precipitate was suspended with 20 mM Tris–HCl (pH 8.0) followed by sonication on the condition of 5s-on, 7s-off for 10 min. After that, the protein samples from both the supernatant and the precipitant were collected again by centrifugation at 8000 rpm for 10 min at 4°C and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).After the same amount of protein samples were separated by SDS-PAGE, the unstained gels were electro-transferred onto immune-blot polyvinylidene difluoride (PVDF) membranes . Following blocked with 5% skim milk in PBST buffer for 1 h, an anti-his antibody (TransGen Biotech) was added at a 1:2000 dilution and incubated overnight. After washed three times with PBST buffer, the PVDF membrane was treated with goat anti-mouse IgG alkaline phosphatase-conjugated secondary antibody at a dilution of 1:1000 (Bio-Rad) for 2 h and washed again. The position of the bound antibodies on the membrane was detected by submerging it in a solution containing nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), following a standard protocol.lacZ reporter gene was used for assaying the transcriptional levels. A series of nested fragments from yneP promoter region were fused to a promoter-less lacZ gene in pIS284. The truncated promoter regions were digested with HindIII and EcoRI, and then inserted into the similarly digested pIs284 plasmid. Finally, the successfully constructed plasmids, including BSP1, BSP2, BSP3, BSP4, BSP5, BSP6, BNP1, BNP2, BNP3, BNP4, BNP5, and BNP6, were transformed into B. subtilis 168 or B. nematocida B16, respectively. After the bacterial strains were cultured in LB medium for 30 h, β-galactosidase activities were determined with GENMED-galactosidase assay kit.Plasmid pIs284 that contained a A headspace solid-phase microextraction (SPME) method in combination with GC-MS was employed to detect methylketones, including 2-heptanone, in different bacterial cultures. The methods of sample preparation and detection were similar to our previous reports . BrieflyB. nematocida B16, B. subtilis 168, E. coli BL21 with heterologously expressed YneP and YtpA, and B. subtilis 168 overexpressing YneP’.The bacterial strains for SPME-GC-MS assay include The total RNA was isolated using an RNA extracting kit  following the treatment of DNaseI to avoid DNA contamination. RNAclean Kit  was then employed to further purify the total RNA. RNA concentration was determined by measuring absorbance at 260 nm using a UV spectrophotometer. After random-primed cDNAs were generated, qPCR analysis was performed with SYBR Green JumpStart Taq Ready Mix for qPCR kit (Sigma-Aldrich Co.) following manufacturer’s instructions. The partial sequence of 16S rRNA was used as an internal control. The PCR amplification used 40 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 40 s on ABI PRISM 7000 Real-Time PCR. The real-time PCR experiments were repeated three times for each reaction using independent RNA sample.The test bacterial strains were grown overnight at 37°C with 200 rpm shaking in 3 ml YPD (Yeast Extract/Peptone/Dextrose) medium. Then each 100 μl bacterial culture was diluted with M9 medium to 600 μl and transferred into 16-well plates. Seeded with L4 stage hermaphrodite nematodes (30–60 per well), the infection assay was performed at 25°C for different time points. Mortalities of nematodes were defined as the ratio of dead nematodes over the total number of tested nematodes. All infection experiments were performed in triplicates and were repeated at least three times.E. coli P50) were at the attractant source and the counter-attractant source, respectively. At the same time, 1 μl sodium azide with the concentration of 1 M was placed to anesthetize animals within about a 0.5 cm radius of the attractant or counter-attractant. After the tested worms of C. elegans were placed near the plate center, the numbers of animals at the attractant and the counter-attractant areas were determined within 1 h. The chemotaxis index was calculated as /total animal number.Chemotaxis assay was performed based on the modified method reported previously . In the t test.All the data were expressed as the mean ± standard deviation (SD). Statistical comparisons were performed by a one-way analysis of variance (ANOVA) followed by Dunnet-shMKSI (AY701574) and shMKSII (EU883793) from wild tomatoes with the annotated genome of B. nematocida B16 at amino acid level, and two genes of ytpA and yneP were identified as having the highest sequence identity to shMKSI and shMKSII. The sequences of ytpA and yneP were then submitted in GenBank with accession number of MH687930 and MH687929, respectively. Compared to the 265-amino acid shMKSI and the 149-amino acid shMKSII, the full ORF of ytpA was 777 bp and encoded 259 amino acids, which was annotated as a putative lysophospholipase. The other gene yneP was 414 bp long and encoded a 138-amino acid hypothetical protein with unknown function. Between them, YtpA shared 31% identity and 54% positive amino acid sequence with shMKSI . Thus, heterologous expressions of those two genes were further carried out in E. coli BL21, and the changes of methylketone production were determined using SPME-GC-MS analysis. First, the SDS-PAGE analysis confirmed that the recombinant proteins of YtpA and YneP were expressed and had the molecular weight of 29.5 and 16.1 kDa as expected  which is one of the intermediate products in fatty acid synthesis  and β-ketoacyl-ACP synthase III (fabH), two key enzymes in the de novo fatty acid synthesis, were determined again under the above carbon sources using qPCR. Between them, FabH catalyzes the quintessential ketoacyl synthase reaction with malonyl ACP and acetyl CoA, and FabF catalyzes the related reaction. As expected, the relative mRNA levels of fabH and fabF in glycerol-contained medium were the highest among the three media (followed by glucose and ethanol) and increased by about two or fourfolds within 2 h, respectively  was achieved in B. subtilis 168. Production of the target protein was validated by Western blot using anti-his antibody  and transformed into their host bacterial strains, β-galactosidase activity of each strain was measured when cultured in LB medium for 30 h. Our data showed that most of the cis-acting elements analyzed in B. subtilis 168 positively regulated the expression of yneP’ except for DegU (yneP promoter of B. nematocida B16, TnrA and MntR negatively regulated yneP expression, but CodY, ComK, and SinR had positive effects (yneP’ in B. subtilis 168 was not more strongly suppressed than B. nematocida B16.The B16 yneP . After afor DegU . To the  effects . FurtherB. subtilis 168, we scanned each peak of GC-MS again and found a peak corresponding to 6-methyl-2-heptanone detected in both B. subtilis 168 and B. subtilis 168 EyneP’ (B. nematocida B16. Our results suggest that 2-heptanone synthesized by the methylketone synthase YneP’ could be methylated and transformed into 6-methyl-2-heptanone upon its production in B. subtilis 168.To further understand the reason for the lack of 2-heptanone in 8 EyneP’ , but no shMKSI and shMKSII are identified to be responsible for the production of methylketones during fatty acid biosynthesis (shMKSI gene is epistatic to shMKSII in the pathway for methylketone biosynthesis. MKS homologs have also been found in other plants and non-plant organisms (B. nematocida B16, ytpA and yneP were identified as the homolog of shMKSI and shMKSII, respectively. The yneP gene was predicted as an unknown thioesterase, which belonged to a large group of hydrolytic enzyme superfamily with a “hot-dog”-like structure. Its activity as a methylketone synthase was verified when the methylketones with medium chain length were obtained in yneP-overexpressed E. coli but absence in the negative control. Furthermore, the data from transcriptional analyses also supported that the methylketones in B. nematocida B16 were synthesized during de novo fatty acid synthesis rather than via the degradation pathway. The other YtpA gene, though homologous with the tomato shMKSI, showed no obvious contribution to methylketone production. Therefore, in Bacillus sp., we hypothesize that yneP likely catalyzes β-ketoacyl-A, the intermediates of fat acid synthesis, into 3-ketoacids which are then further decarboxylated spontaneously. But the role of YtpA, the homolog of shMKSII in B. nematocida B16, remains to be confirmed.The prevalence of methylketones with an odd number of carbons in nature suggests that they are most likely derived from decarboxylation of the corresponding β-ketoacids that are intermediates in either the biosynthesis or the degradation pathways of fatty acids. Of the two pathways, the spontaneous decarboxylation is more rationally expected since CoA esters and free β-ketoacids, the intermediates in fatty acid degradation are unstable. Wild tomatoes produce methylketones as repellants against its pests , in whicynthesis . In thisynthesis . So the rganisms . HoweverKodamaea ohmeri, carried by honeybees can produce more abundant 2-heptanone using the pollen collected in honeycomb, which attracts members of the intruder Aethina tumida and leads to disastrous damages for honeybees (B. nematocida B16 strain can successfully lure the worms and fulfill one of the prerequisites for a Trojan horse-like infection (B. subtilis 168 showed little attraction for nematodes, even though the gene yneP’ had methylketone synthetase activity. The similar expressional levels of the homologs between those two bacteria form stark contrast to their vast differences in both 2-heptanone production and attractive capability. Our data further suggest that instead of producing 2-heptanone as a final product, 2-heptanone was likely converted into 6-methyl-2-heptanone in B. subtilis 168. This metabolic transformation may be an effective strategy to prevent the non-pathogenic B. subtilis 168 from being the food of nematodes. Our results provided a co-evolution paradigm of second metabolites during the interactions between pathogenic/non-pathogenic bacteria and host.In nature, 2-heptanone functions as an important allomone in the interaction between prey and predator. For example, when worker bees attack their enemies by sting, they often use the upper jaw to bite the enemy. Synchronously, the chemicals, such as 2-heptanone, are left on the enemy’s bodies to guide the other bees for attacking . Furtheroneybees . Interesnfection . But theXH and KZ designed the research. MZ, XX, YL, and PW carried out the experiments. KZ and SN analyzed the data. XH and MZ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Current evidence suggests that some of the most effective weight loss approaches are changes in dietary and physical activity behaviors through lifestyle modification programs. The Group Lifestyle Balance (GLB) program is a group-based behavior modification program aimed at changing diet and physical activity for weight loss. It was developed to be more cost-effective and easier to disseminate than its individually administered parent program, the Diabetes Prevention Program (DPP). However, the average weight loss following participation in the GLB is only approximately 3.5%, with low long-term weight loss maintenance.We aimed to optimize the weight loss outcomes of the GLB to increase the efficacy already afforded by its cost-effectiveness and ease of dissemination. We did this by integrating the habit formation tool of if-then plans into the program. This program is called the enriched GLB or the McGill Comprehensive Health Improvement (CHIP) Healthy Weight Program. Results at 3 and 12 months of participation have already been published elsewhere. They showed no between-group differences between the standard and enriched GLB but higher weight loss in both groups compared to the DPP. This paper reports the long-term weight loss maintenance data following participation in the program.Of the 172 participants enrolled at the beginning of the study, data from 110 participants were available and analyzed at 24 months, i.e., 12 months after the end of the 12-month intervention.No between-group difference in weight loss maintenance was observed. Pooled results showed a significant weight regain from 12 to 24 months, i.e., an average of 7.85 lbs. of the 20.36 lbs. lost. However, participants from both groups were still 12.51lbs or 6.13% lighter at 24 months than at baseline.If-then plans did not result in a higher percentage of weight loss at 24-month follow-up compared to the standard GLB. However, at 24 months, both groups did show a maintenance of a significant portion of the weight lost at the end of intervention.NCT02008435, registered 6 December 2013.ClinicalTrials.gov Identifier:  Behavior modification programs aimed at changing diet and physical activity have been shown to be an effective weight loss approach . The mosDue to the high cost of the DPP, a group-based shortened version, called the Group Lifestyle Balance (GLB) program, was developed . While lTo increase the weight maintenance effectiveness of the GLB, our study integrated habit formation techniques, specifically if-then plans, to reinforce habit change , 9. We cgroup participants, just more implicitly. Also, the coaches in both groups were highly trained in cognitive behavioral therapy (CBT) [The GLB standard and enriched program versions were delivered over 1 year . The 3- (end of core sessions) and 12- (end of intervention) month results of the if-then enriched randomized controlled trial (RCT) have been published . No betwpy (CBT)  and spenThis paper reports the findings of our RCT after a 1-year no-contact follow-up period, 24-months after baseline. We hypothesized that the if-then plans would serve as a protective barrier to weight regain. Specifically, we hypothesized that from the end of intervention (12 months) to follow-up (24 months), the if-then plan group would show greater weight loss maintenance than the group that was not explicitly instructed to create if-then plans.The prospective, two-arm RCT was conducted between 2014 and 2017 with approval by the Research Ethics Board at McGill University . A detailed description of the intervention, methods, procedures, and measures are published in the study protocol  and outlThe GLB manual was adhered to in both groups , and if-The primary outcome, body weight, was assessed using a digital scale. Details of the measures of the secondary outcomes of goal achievement, diabetes risk factors, physical activity, self-monitoring, and habit strength are available in the study protocol . Self-moN = 62 participants were handled with the estimation procedure “use full information maximum likelihood” with robust standard errors. This allows all data to be included in the estimation [p = 0.608), the missingness was assumed to follow a missing completely at random (MCAR) pattern. The full information maximum likelihood method performs equally well as listwise (or pairwise) deletion under MCAR [α = .05 indicated no group difference in the estimate examined. Due to the nonsignificant model difference, average pooled change scores were computed across groups.Study analyses were conducted using Mplus version 8.0 . Multigrtimation , 16. As Demographic information of the participants at baseline and 24-month follow-up is presented in Table p = .047), with participants regaining on average 7.85 lbs. of the 20.36 lbs. that they had lost. However, participants were still 12.51 lbs. (or 6.13%) lighter at 24 months than at baseline; they had lost 9.98% of their initial body weight at the 12-month follow-up. More than half of all participants who achieved a clinically significant weight loss of 5% at post-intervention (62.07%) maintained this weight loss at 24 months.Table Pooled results showed significant changes at 24-month follow-up only for self-monitoring and habit strength. All other variables showed no significant change from 12 to 24 months; that is, all changes achieved at 12 months were maintained.Food tracking frequency significantly decreased for both groups from 12 months to 24 months by 0.49 days/week. Activity tracking frequency also decreased for both groups by 1.10 days/week. In comparison to their baseline values of 5 days/week for food tracking and 6 days/week for activity tracking frequency, both values were significantly lower at 24 months.Habit strength showed a statistically significant decrease from 12 to 24 months by a mean score of 0.44. However, it remained significantly higher than the baseline total score of 2.This paper reports the 24-month follow-up results of an intervention developed to increase and maintain weight loss of the GLB. Both groups showed long-term weight loss of about 6.13% compared to baseline, even though some weight regain occurred post-intervention. A weight loss of above 5% is considered to be clinically significant indicating an effective behavioral weight loss program [Pooled results over both conditions show that the long-term maintenance for our program was slightly better than the long-term maintenance in Piatt et al.’s study , which iA few limitations of the current study should be mentioned. Our 24-month results are compared only with one other study, namely that of Piatt et al. . It is tThe attrition rate at follow-up, which was 46% in this study (107 to 51 participants in the enriched GLB group and 101 to 59 in the standard GLB group), was another limitation of this study. However, the degree of attrition is typical for long-term follow-ups , and ourThe current study has implications for ways to achieve better long-term weight loss maintenance in group-based behavioral weight loss programs. Specifically, our study draws attention to the importance of training coaches to effectively teach behavior change techniques to participants. These techniques include if-then contingencies, which are an integral part of behavior planning and habit formation.We found large reductions in weight from baseline to 3 and 12 months for both the standard and the enriched GLB, with a significant portion maintained at 24 months. Participants lost 9.98% of their initial body weight at 12-month follow-up and retained this weight loss at 6.13% of their initial baseline weight at the 24-month follow-up. Furthermore, a greater percentage of those who lost 5% of their initial weight at the end of intervention maintained this weight loss at 24-month follow-up than in the most comparable study assessing weight loss maintenance of the standard GLB.


In-situ pH-sensitive cellulose fibers (IS-pH-SCF) were prepared by anchoring bromothymol blue (BTB) onto cellulose fibers (CF) modified with hydroxypropyltriethylamine (HPTTL) groups. Fourier transform infrared and X-ray photoelectron spectrum analyses demonstrated that the HPTTL groups were grafted onto the CF. X-ray diffraction proved that cellulose I in the CF transformed into cellulose II after quaternization. Scanning electron microscopy suggested that the quaternized CF (QCF) surface was clean and uniformly ridged. The adsorption of BTB onto QCF was carried out via batch adsorption experiments. A kinetic study illustrated that the adsorption was a spontaneous process and described well by pseudo-second-order, Freundlich and Temkin isotherms. The activation energy for the BTB adsorption onto QCF was 52.89 kJ/mol, which proved that the BTB adsorption onto QCFs was chemically controlled. The pH response demonstrated that the IS-pH-SCF was highly sensitive to pH, with an obvious color change for pH 4 to 8. The release tests showed that BTB was anchored on QCFs and that no BTB was released. IS-pH-SCF has a potential use for indicating pH changes in food. Foods are rich in nutrients and are the most basic materials for human survival. Food safety is directly related to human physical health and life safety, and is critical to ensure both economic and social stability. Food quality deteriorates because of moisture, light, microbial reproduction during transportation, and storage. Food spoilage could lead to a reduction or loss of nutritional value and even cause food poisoning, with a potential for great harm and significant economic losses. Improvements in living standards have resulted in people paying increased attention to food safety. The traditional food quality test is based on a chemical analysis, which requires the destruction of foods, expensive analytical instruments, professional operators, and extended times . Such and-glucopyranosyl units [Bromothymol blue (BTB) is a pH-sensitive synthetic dye that is used extensively in indicators ,24,25. Ayl units . CF is iyl units . It is aIn this study, CFs were positively charged via a cationic modification in which quaternary ammonium groups were grafted onto cellulose molecules. In-situ pH-sensitive CFs (IS-pH-SCF) were prepared by anchoring BTB onto quaternized CFs (QCF) in a NaOH solution. The QCF and IS-pH-SCF were analyzed by Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and scanning electron microscopy (SEM). QCF was tested as an adsorbent for BTB from a NaOH solution. The effects of the QCF dosage and contact time were investigated. The kinetics and isotherms of the adsorption process were studied. The pH-sensitivity of the IS-pH-SCF and the non-release of BTB were measured.Bleached softwood kraft pulp was used as a cellulose fiber (CF) and offered by Henfeng Paper Co., Ltd. . Bromothymol blue (BTB) was supplied by the Guangfu Chemical Research Institute . All other chemicals, including sodium hydroxide, epoxy chloropropane, triethylamine, hydrochloric acid, absolute ethyl alcohol and buffer solutions (pH 4 to 8), were supplied by Yongda Chemical Reagent Co., Ltd. . All chemicals were of analytical grade and used without purification. w/w) were stirred at 500 rpm and 25 °C for 2 h. A NaOH solution  and 60 mL of epoxy chloropropane were added into the flask after the solution had been removed by filtration. The mixture was heated to 65 °C and stirred at 500 rpm for 7 h. 70 mL of triethylamine/absolute ethyl alcohol solution (34% v/v) was added into the flask after a solid-liquid separation, and the mixture was stirred at 500 rpm and 75 °C for 4 h. Absolute ethanol was used to wash the resulting product to remove the unreacted triethylamine. Then, the product was washed successively with 0.1 M NaOH, 0.1 M HCl and distilled water until it reached a pH of 7. The QCF was dried at 65 °C for 10 h.QCF was prepared according to the literature with slight modifications . The CF −1 with a resolution of 4 cm−1. An XPS analyzer with K-Alpha  was employed to detect the X-ray photoelectron spectroscopy. The XRD patterns of the CF and the QCF were acquired by using a D/MAX-2500 diffractometer  at 1200 W . A Hitachi SU-70 microscope  was employed to analyze the surface morphology of the CF and the QCF. A thin layer of gold was deposited on the surface of the samples before the observation.The FTIR spectra of the CF and the QCF were measured by using a Nicolet 6700 spectrometer , and tests were conducted from 500 to 4000 cmt, mg/g) and the equilibrium  are obtained from the following equations:0, Ce and Ct (mg/L) denote the concentration of the BTB/NaOH solution at initial, equilibrium and t (min), respectively. V (L) represents the volume of BTB/NaOH solution and m (g) presents the QCF dosage.The adsorption of BTB on the QCF in the dilute alkaline solution (0.1 M NaOH) and the effects of both the QCF dosage and the adsorption time on the adsorption were investigated by batch adsorption experiments. The standard curve of BTB in a 0.1 M NaOH solution was obtained by using an ultraviolet–visible spectrophotometer . For each adsorption experiment, 50 mL of BTB/NaOH solution with 1000 mg/L and a certain amount of QCF were added into a beaker under magnetic stirring of 200 rpm for different times until the adsorption reached an equilibrium. The mixture was separated by using a benchtop high-speed centrifuge  at 10,000 r/min for 6 min. Subsequently, the residual BTB/NaOH solution was diluted 50 times with a 0.1 M NaOH solution, and then the absorbance was measured with the ultraviolet–visible spectrophotometer at 615 nm. The concentration of BTB was calculated according to the standard curve. The adsorption capacity at time t (qe and qt (mg/g) indicate the quality of BTB adsorbed onto the QCF at equilibrium and t (min), respectively. The qe1 (mg/g) denotes the theoretical value of BTB adsorbed onto the QCF at equilibrium, and K1 (1/min) represents the rate constant.The adsorption experiments were performed by magnetically stirring 100 mg of QCF and 50 mL of BTB/NaOH solution with 1000 mg/L at 200 rpm for different time intervals at various temperatures to explore the kinetics. The adsorption process was analyzed via pseudo-first-order and pseudo-second-order models . In genee2 (mg/g) denotes the theoretical quality of BTB adsorbed onto the adsorbent at equilibrium, and qt (mg/g) is denotes the theoretical quality of BTB adsorbed onto the adsorbent at time t (min). The K2 (g/mg min) represents the rate constant.The pseudo-second-order model describes the whole process of the adsorption and conforms to the mechanism of chemical adsorption . The modThe activation energy Ea  of the am (mg/g) is the maximum adsorption capacity, qe (mg/g) is the equilibrium adsorption capacity, Ce (mg/L) represents the concentration of the BTB/NaOH solution at equilibrium, and KL (L/mg) denotes the Langmuir adsorption constant. Kf denotes the Freundlich constant and n denotes the heterogeneity factor. A and B are the Temkin dasorption isothermal constants [50 mL of a BTB/NaOH solution with 1000 mg/L and a certain quality  of QCF were stirred in a magnetic stirring water bath at 200 rpm for 4 h at different temperatures  to study the adsorption isotherm. Langmuir  and Temkonstants .The QCF (2 g) and 200 mL of a BTB/NaOH solution (1000 mg/L) were added into a beaker and stirred in a magnetic stirring water bath at 200 rpm for 4 h at 50 °C. The mixture was separated via filtration with a 200 mesh filter cloth. The isolated solids were soaked in distilled water and stirred at ~500 rpm for 5 min, followed by a vacuum filtration, and this was repeated 5 times until the filtrate was colorless and neutral. 0.1 g of IS-pH-SCFs and distilled water (50 mL) were stirred at 500 rpm to disperse the IS-pH-SCFs uniformly. Following this, the suspension was added slowly into the Buchner funnel equipped with two layers of filter paper and vacuum filtration was performed at 0.08 MPa. Circular IS-pH-SCF felts were obtained and dried at 65 °C.The FTIR analysis and SEM observation of IS-pH-SCF were conducted by using the same methods as those for QCF.0, a0 and b0 are chromatic values of the IS-pH-SCF. L, and a and b are chromatic parameters of the IS-pH-SCF at different pH values.The circular pieces of IS-pH-SCF felts were cut into rectangles (2 cm × 2 cm). Individual buffered solutions  were placed in glass culture dishes (marked 4 to 8). The IS-pH-SCF felt was soaked in a buffer solution for 30 s, then removed with tweezers and placed on several layers of filter paper. A portable colorimeter  was employed to analyze the color of the IS-pH-SCF rectangles, including L (luminosity), a (carmine-green) and b (yellow-blue). Five detections were performed for each IS-pH-SCF. The total chromatic difference (∆E) was obtained as:A piece of the IS-pH-SCF was soaked in distilled water (pH = 7) for 24 h, before the IS-pH-SCF was taken out with a pair of tweezers. Subsequently, the color of the resulting distilled water was observed after the very small fibers were separated by centrifugation. An HCl solution (0.1 M) was dropped on an IS-pH-SCF felt on a piece of filter paper, and the color change of the IS-pH-SCF was observed. After 5 min, the IS-pH-SCF was removed and the color of the original location where IS-pH-SCF had been was observed before and after drying. In addition, the experiments were also conducted by using a NaOH solution (0.1 M).−1 is the O–H stretching vibration, and the band at ~2906 cm−1 corresponds to the C–H stretching of the –CH2– groups. The bands at ~1158, 1098, 1057, and 1030 cm−1 originated from the C–O–C stretching from the glycosidic bonds of the cellulose molecule [−1, corresponding to the C–N stretching vibration of –N+(C2H5)3 [+(C2H5)3 has been grafted onto the cellulose skeleton. In the XPS spectrum (molecule . After q+(C2H5)3 . The chaspectrum B, peaks The effects of the adsorption time on the BTB adsorption onto the QCF at 303.15, 313.15 and 323.15 K are exhibited in As can be seen from e  and experimental qe  values was large. This disparity showed that the adsorption of BTB on the QCF cannot be accurately described with the pseudo-first-order kinetic model. The R2 of the pseudo-second-order at three experimental temperatures were 0.998, 0.997 and 0.997, which indicated excellent linearity. The difference between the calculated qe  and the experimental qe  is smaller. Therefore, the pseudo-second-order can describe the adsorption process well.Pseudo-first-order  and pseuThe activation energy of adsorption was calculated from the Arrhenius equation. In general, the values of the activation energy can be applied to distinguish whether the adsorption is physically (4~40 kJ/mol) or chemically controlled (higher than 40 kJ/mol) . The actm and KL are negative, which indicated that the adsorption of BTB onto the QCF did not fit the Langmuir model well. However, the values of the correlation coefficient (R2) were higher than 0.96 for the Freundlich and Temkin isotherm models, which described the BTB adsorption onto the QCF well. Adsorption isotherms can tell us how the adsorption conducts and help us analyze the interaction between the adsorbate and the adsorbent ,44. Lang−1 was ascribed to the O–H. The band at ~2880 cm−1 was attributed to the C–H for –CH2– groups. The bands at ~1158, 1098, 1057, and 1030 cm−1 corresponded to the C–O–C from the glycosidic bonds of the cellulose molecule. After adsorption, the strength of the O–H band was enhanced and moved to a low wavenumber. The band at 2880 cm−1 became stronger, which indicated that the new –CH2– groups had been introduced into the QCF structure. A new peak at ~1330 cm−1 indicated the S=O stretching, and showed that the BTB had been included in the IS-pH-SCF structure. From the adsorption experiments, the maximum adsorption capacity was 38 mg/g for 1000 mg/L of BTB/NaOH solution and a dosage of 100 mg for 4 h. The BTB content of IS-pH-SCF is 0.038% (w/w).The SEM photographs in The response of the IS-pH-SCF at different pH values was shown in The release of BTB from IS-pH-SCF in distilled water and dropping acid or alkaline solutions was investigated. As shown in IS-pH-SCF was prepared by anchoring BTB molecules on a cationic cellulose fiber through adsorption. It was proven by FTIR and XPS analyses that the cationic modification by grafting hydroxypropyltriethylamine groups onto CFs was completed. SEM observations showed that the QCF surface became uniformly wrinkled. QCF was used as an adsorbent to anchor BTB in the alkaline solution. A higher temperature favored BTB adsorption onto the QCF. The maximum adsorption capacity was 38 mg/g for 1000 mg/L of BTB/NaOH solution and a dosage of 100 mg for 4 h. The adsorption of BTB on the QCF followed pseudo-second-order kinetics, and Freundlich and Temkin isotherms. The IS-pH-SCF exhibited a different color for each pH value from 4 to 8. Simultaneously, the BTB was not released during the pH response, which conformed that the BTB was anchored firmly on the cationic cellulose fiber. This study provides a new path for fabricating a pH-sensitive fiber that could be used to monitor the freshness of food without any pollution.


Treating cerebral ischemia continues to be a clinical challenge. Studies have shown that the neurovascular unit (NVU), as the central structural basis, plays a key role in cerebral ischemia. Here, we report that anthocyanin, a safe and natural antioxidant, could inhibit apoptosis and inflammation to protect NVU in rats impaired by middle cerebral artery occlusion/reperfusion (MCAO/R). Administration of anthocyanin significantly reduced infarct volume and neurological scores in MCAO/R rats. Anthocyanin could also markedly ameliorate cerebral edema and reduce the concentration of Evans blue (EB) by inhibiting MMP-9. Moreover, anthocyanin alleviated apoptotic injury resulting from MCAO/R through the regulation of Bcl-2 family proteins. The levels of inflammation-related molecules including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), which were over-expressed with MCAO/R, were decreased by anthocyanin. In addition, Nuclear factor-kappa B (NF-κB) and the NLRP3 inflammasome pathway might be involved in the anti-inflammatory effect of anthocyanin. In conclusion, anthocyanin could protect the NVU through multiple pathways, and play a protective role in cerebral ischemia/reperfusion injury. The use the BBB .Apoptosis and the inflammatory response are important processes that cause NVU destruction, and they play a key role in the pathology of stroke . In stroAnthocyanins are natural flavonoids formed by the combination of anthocyanidin and glycosides, which are widely found in the cytoplasm of flowers, fruits, stems, leaves, and root organs of plants. Studies have confirmed the biological benefits of dietary anthocyanins, as they exhibit anticancer activity, anti-inflammatory activity, neuroprotective activity, anti-obesity activity, anti-diabetic activity, and properties involved in the prevention of cardiovascular disease . AnthocyIn this study, we investigated the protective effects of anthocyanin in rats with MCAO/R injury, and found some clues on the underlying mechanism of protection.All procedures described in this study were performed in accordance with the guidelines of Nanjing University of Chinese Medicine Animal Care and Use Committee.Chromatographic grade methanol and acetonitrile were purchased from Merck . Formic acid, ethyl acetate and other chemicals were purchased from commercial sources and of analytical grade. Rabbit monoclonal to Bad, rabbit monoclonal to Bcl-xl, rabbit monoclonal to Bax, rabbit polyclonal to Bcl-2, rabbit polyclonal to p-NF-κB p50, rabbit polyclonal to p-NF-κB p65, and rabbit monoclonal to p-IκB were purchased from Abcam . Antibodies for mouse monoclonal to NLRP3 were acquired from Adipogen International . Antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were acquired from Sigma-Aldrich . Evans blue (EB) was purchased from Sigma Chemical Co., Ltd. . All other reagents were obtained from Sigma-Aldrich .O-rutinoside (p-coumaroyl)-5-O-glucoside (purity 98.3%) was extracted and purified from the dried fruits of Lycium ruthenicum Murr. according to the method proposed by Anthocyanin, namely pentunidin-3-O-rutinoside (p-coumaroyl)-5-O-glucoside was separated from the second fraction of the Sephadex LH-20 eluate by an Alltech 1500 semi-preparative HPLC  and was identified using an AB SCIEX API 4000 ESI-MS/MS .Briefly, anthocyanin was extracted twice from the dried fruits of LRM using 1% formic acid (40°C) at a ratio of 1:30 for 1 h each time. The crude extract of anthocyanins was subjected to liquid–liquid extraction, four times by water-saturated ethyl acetate at a ratio of 1:3 for 12 h each time, and was purified using a column (1 cm × 15 cm) of Amberlite XAD-7HP macroporous adsorption resin . Then, two fractions of anthocyanins were isolated from the Amberlite XAD-7HP eluate using a column (1 cm × 100 cm) of Sephadex LH-20 . Subsequently, the monomeric anthocyanin of pentunidin-3-Adult male Sprague–Dawley rats weighing 250–300 g were provided by Model Animal Research Center of Nanjing University. All animals were housed five per cage under controlled temperature (22 ± 2°C) and with 12 h light/dark cycle (lights on at 8:00 a.m.). The animals had free access to food and water. Rats were injected intraperitoneally with different doses of anthocyanin  for 7 days before the induction of middle cerebral artery occlusion.The common carotid artery was isolated following rat anesthesia , and the branches of the right external carotid artery were carefully separated. A 4-0 nylon monofilament suture with a silicon coated tip was advanced to the internal carotid artery to occlude the source of the MCA. In the sham group, this same procedure was performed but no sutures were inserted. After 2 h of cerebral ischemia, the suture was withdrawn for reperfusion for 24 h.Each rat was evaluated for neurological deficits according to Longa’s method , which iEach rat was evaluated for cognitive function by novel object recognition test, 24 h before the test, the mice were habituated to the arenas (50 cm × 50 cm plastic container) for 5 min without objects. The day after the mice re-entered the arenas from the same starting point of the arena (facing the bottom left corner) and granted 10 min to familiarize themselves with the two identical objects. Exactly 1 h after the familiarization period, the rats are exposed to two different object: one is same with the training phase and another is a novel object. The time that the rats spend to explore the old and novel objects are recorded, calculated the object interaction ratio according to the formula: object interaction ratio = Novel object exploration time/Old object exploration time.After 2 h of MCAO and 24 h of reperfusion, the brain was quickly removed. Infarct volume was then measured by TTC staining, which revealed the severity of cerebral ischemia. The data was expressed as a percentage of the total hemisphere .Brain water content was measured using a dry-wet method. Brain tissue was removed after 24 h of reperfusion, and was immediately weighed (a) It was then baked in a 120°C oven for 24 h and weighed again (b) Brain water content was measured according to the formula: brain water content = (a–b)/a × 100%.The rats in each group were injected with 2% EB (2 ml/kg) in the tail vein 2 h before sacrifice. The brain tissue was then removed and weighed, and then placed into dimethylformamide (1 ml/100 mg brain tissue) and incubated at 60°C. for 24 h. After centrifugation at 1000 r/min for 5 min, the supernatant was taken and the absorbance at a wavelength of 620 nm was measured with a spectrophotometer.Protein concentrations were determined after samples collection. The samples, mixed with loading buffer, were loaded into the wells of a precast gel and subjected to electrophoresis. Then the gel was washed in washing solution followed by shaking in incubation solution for 48 h (37.5°C). After staining with Coomassie Brilliant Blue, the gel was bleached by different concentrations of methanol-acetic acid.Samples of 0.5 ml of blood were obtained from the femoral vein at 24 h after reperfusion. The serum was separated and collected by centrifugation. Then the serum samples were then assessed for inflammatory mediator  levels using an ELISA kit, according to the manufacturer’s instructions.Tissues were placed in a pre-cooled glass grinder, a pre-cooled lysate containing PMSF and phosphatase inhibitors was added, and they were thoroughly ground and lysed. An appropriate amount of 5X protein loading buffer was added to the protein sample. The protein (50 μg) was separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin at room temperature for 1 h and then incubated with a specific primary antibody overnight at 4°C. The following primary antibodies used: Bad (1:2000), Bax (1:1000), Bcl-2 (1:1000), Bcl-xl (1:1000), p-NF-κB p50 (1:1000), p-NF-κB p65 (1:2000), p-IκB (1:5000), and NLRP3 (1:500). The corresponding secondary antibody was incubated for 1 h at room temperature and the blot was visualized using the enhanced chemiluminescence (ECL) method. Software Quantity One -4.6.5 software  was used to analyze the data.Trizol reagent was added to the sample, which was homogenized, and the RNA was extracted. cDNA was then synthesized and SYBR Green I dye was used for real-time quantitative PCR. The sense and antisense primers used for the analysis of rat TNF-α, IL-1β, IL-6, and GAPDH expression were as follows. TNF-a: 5′-CCC CTTTATCGTCTACTCCTC-3′ and 5′-GCTGGTAGTTTAGCTCCGTTT-3′ (553 bp). IL-1b: 5′-TCATTGTGGCTGTGGAGAAG-3′ and 5′-CTATGTCCCGACCATTGCTG-3′ (579 bp). IL-6: 5′-GGATACCACCCACAACAGAC-3′ and 5′-TTGCCGAGTAGACCTCATAG-3′ (520 bp). GAPDH: 5′-CCATCACTGCCACTCAGAAGA-3′ and 5′-CATGAGGTCCACCACCCTGT-3′ (446 bp).After the tissue was formed into a block of wax, it was cut into 4 μm slices, dewaxed, and hydrated. It was stained with hematoxylin for 4 min and eosin for 90 s. It was dehydrated with an alcohol gradient from low to high concentrations, made transparent with xylene, and then observed under the microscope .The terminal deoxy transferase reaction mixture was incubated with the sections for 1 h according to the manufacturer’s instruction. All of the counted nuclei were stained with hematoxylin.p value < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism Version 5.01 software .Statistical analysis was carried out using ANOVA. Results are expressed as mean ± SD. A To evaluate the protective effect of anthocyanin, different concentrations of anthocyanin  were injected intraperitoneally into rats before the MCAO/R surgery. The infarct area of the brain was examined by 2,3,5-Triphenyltetrazolium chloride staining. MCAO/R produced massive infarction, however, in the MCAO/R rats that received anthocyanin injections, the infarct volume was found to be significantly reduced in a dose-dependent manner Figures . AdditioBlood-brain barrier is the core structure of the NVU, which is damaged in MCAO/R. Therefore, we next examined brain edema and leakage, which is believed to reflect the permeability of the BBB. The results show that cerebral edema in the anthocyanin-treated rats was significantly alleviated compared with MCAO/R rats Figure . SimilarMany studies have confirmed that MMP-9 causes BBB damage in cerebral ischemia . TherefoMany studies have reported that the Bcl-2 family is an important apoptosis regulator in the mitochondria . A largeNext, we examined the effect of anthocyanin on the inflammatory response. The results showed that compared with the sham rats, MCAO/R significantly up-regulated the expression of TNF-α, IL-1β, and IL-6 in the cerebral cortex, and anthocyanin significantly inhibited the expression of TNF-α, IL-1β, and IL-6 Figure . We thenEvidence shows that NF-κB and the NLRP3 inflammasome pathway play an important role in the inflammatory signaling pathways . To idenOur study demonstrated that the administration of anthocyanin significantly reduced infarct volume, neurological scores, cognitive function decline, cerebral edema and the concentration of EB in MCAO/R rats by inhibiting MMP-9, Bcl-2 family and inflammation-related molecules including TNF-α, IL-1β, and IL-6. In addition, NF-κB and the NLRP3 inflammasome pathway may play a role in the anti-inflammatory effect of anthocyanin. Taken together, these results may enhance our understanding of the NVU protection properties of anthocyanin against ischemic stroke through multiple pathways and provide an alternative option for the future prevention and treatment of stroke.Stroke is one of the most common causes of death worldwide and is a major cause of acquired disability in adults, with highly complex pathological process . In recePrevious studies have shown that MMP-9 is abnormally expressed in brains suffering from cerebral ischemic injury and that it promotes brain damage and BBB breakdown . In humaBlood-brain barrier dysfunction caused by apoptosis and inflammation, is a key factor that causes NVU injury, which promotes the development of ischemic brain injury . ApoptosAfter stroke, cerebral ischemia injury induces a robust inflammatory response, causing great damage to the BBB. Damage-associated molecular patterns (DAMPs) are the main signals that trigger inflammatory reactions. Most DAMPs, including HMGB-1 or heat shock proteins, are released from dying and dead cells , and are−/− mice exhibited reduced infarct volumes, decreased edema formation, and preserved BBB permeability (One study found that the expression of NLRP3 was increased in the brain tissue of stroke patients . The acteability . In thisAnthocyanins, which are flavonoids formed by anthocyanidins and sugars through glycosidic bonds, are natural antioxidants with a wide range of biomedical functions. Anthocyanins are thought to benefit cardiovascular, inflammatory, and degenerative diseases . Anthocy50 for a combination of the glycosides of cyanidin, delphinidin, petundin, and malvidin was more than 25 g/kg for mice and more than 20 g/kg for rats (Although anthocyanins have considerable beneficial effects, we still need to pay attention to the safety of anthocyanins. Anthocyanins do not appear to be readily absorbed or metabolized. A weight-of-evidence analysis indicates that anthocyanins are not genotoxic . The acufor rats . Howeverfor rats . DespiteIn addition, the anthocyanin attenuated progression of cerebral ischemic damage in rats was, associated with the suppression of the JNK and p53 signaling pathway . Our stuTaken together, the NVU is important in stroke pathophysiology, and the investigation of multiple-pathways that protect of the NVU may lead to novel approaches for this devastating disease. Our study found that anthocyanin could inhibit apoptosis and inflammation to protect the NVU in rats impaired by MCAO/R. Additionally, NF-κB, MMP-9, and the NLRP3 inflammasome pathway might be involved anthocyanin’s protective effects in the NVU. Therefore, anthocyanin could protect the NVU through multiple pathways, and might be a potential candidate for the prevention and treatment of stroke.Data and materials will be available from the authors upon reasonable request and with the permission of ZP after submission of the thesis.in vivo experiments. TY helped with the design of the study. YL conducted the RT-PCR experiment and assisted with the manuscript preparation. TJ performed the Western Blot experiment and assisted with the data analysis. YP helped conduct the experiments and contributed to the writing of the manuscript.ZP carried out the experiments and assisted with data analysis. MC and GD helped with the manuscript preparation and the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.